Optical system for chemical and/or biochemical reactions

ABSTRACT

An apparatus for detecting light emanating from chemical or biochemical reactions occurring in at least one reaction vessel of a plurality of reaction vessels is disclosed. Each reaction vessel has a receptacle portion having an emitting area from which light can emanate. A plurality of light waveguides are arranged to guide light from apertures in a masking element to a light dispersing device for dispersing the light from each waveguide into a dispersed spectrum. A light detecting device detects specific spectra in the dispersed spectra of light substantially simultaneously In one embodiment, the light waveguides have a diameter that tapers from a first end substantially similar in diameter to the area of the top of the reaction vessel to a second end that is substantially smaller in diameter.

The present invention relates to an optical system for monitoringreactions, in particular, though not exclusively, for monitoring lightemanating from reaction vessels in which chemical or biochemicalreactions are carried out.

Many chemical and biochemical reactions are carried out which produce adetectable light signal, such as a fluorescent, chemiluminescent orbioluminescent signal, which occurs or is modified under certainreaction conditions. Such signals may emanate due to the reagents orresults of the reaction(s) emitting light under certain conditions, forexample due to excitation energy being applied, or may emanate by beinggenerated by the reaction itself.

Detection of these light signals may be used in a variety of ways. Inparticular they can allow for the detection of the occurrence of areaction, which may be indicative of the presence or absence of aparticular reagent in a test sample, or to provide information about theprogress or kinetics of a particular reaction. Although the term “light”is generally used to include visible light, it will be appreciated thatoptical signals that can emanate from reactions and be detected may alsooccur in the infra-red and/or ultra-violet portions of the spectrum andit is intended that the term “light” encompass all optical signals thatcan emanate from reactions of whatever wavelength that can be detected.

In many instances a reaction mixture may contain more than one“signaling” reagent, and the light signals may need to be detected ormonitored over time, in order to provide a full set of information aboutthe occurrence, nature or progress of a particular reaction.

A particular example of a reaction where detectable signals and inparticular fluorescent signals are monitored is in nucleic acidamplification techniques and in particular the polymerase chain reaction(PCR). Amplification of DNA by polymerase chain reaction (PCR) is atechnique fundamental to molecular biology. PCR is a widely used andeffective technique for detecting the presence of specific nucleic acidswithin a sample, even where the relative amounts of the target nucleicacid is low. Thus it is useful in a wide variety of fields, includingdiagnostics and detection as well as in research.

Nucleic acid analysis by PCR requires sample preparation, amplification,and product analysis. Although these steps are usually performedsequentially, amplification and analysis can occur simultaneously.

In the course of the PCR, a specific target nucleic acid is amplified bya series of reiterations of a cycle of steps in which nucleic acidspresent in the reaction mixture are denatured at relatively hightemperatures, for example at 95° C. (denaturation), then the reactionmixture is cooled to a temperature at which short oligonucleotideprimers bind to the single stranded target nucleic acid, for example at55° C. (annealing). Thereafter, the primers are extended using apolymerase enzyme, for example at 72° C. (extension), so that theoriginal nucleic acid sequence has been replicated. Repeated cycles ofdenaturation, annealing and extension result in the exponential increasein the amount of target nucleic acid present in the sample.

DNA dyes or fluorescent probes can be added to the PCR mixture beforeamplification and used to analyse the progress of the PCR duringamplification. These kinetic measurements allow for the possibility thatthe amount of nucleic acid present in the original sample can bequantitated.

In some systems, sample analysis occurs concurrently with amplificationin the same tube within the same instrument. This combined approachdecreases sample handling, saves time, and greatly reduces the risk ofproduct contamination for subsequent reactions, as there is no need toremove the samples from their closed containers for further analysis.The concept of combining amplification with product analysis has becomeknown as “real time” PCR.

However, the fact that these systems produce complex and oftenoverlapping signals, from multiple different fluorophores within thesystem means that complex signal resolution is required to determine theintensity of the signal from the individual fluorophores.

The complexity is further compounded in that PCRs are generallyconducted in specifically constructed thermal cyclers, such as blockheaters, which accommodate arrays of multiple reaction vessels at thesame time. These are then cycled together, and the signals produced byeach vessel monitored.

Current systems for PCR fluorimetry often rely on detection systems suchas monochrome detectors (CCD, photodiode, PMT, CMOS detectors etc.)which on their own will only detect the presence or absence of light,but cannot distinguish amongst light of different wavebands or colours.Therefore they are not able directly to differentiate between thevarious different fluorophore signals. This problem is often addressedby having an external means of separating or filtering light intodifferent wavebands for detection at different points on the detector,or at different points in time.

These external means increase the cost, size and complexity of theinstrument. Such external means often need to be precisely mounted foroptical alignment, and this tends to reduce the robustness of theinstrument or leads to increased size, weight and cost associated withthe mounting.

Many useful applications of PCR analysis rely on readings from multiplewavebands, and all require each vessel used to be measured; so if theoptical apparatus of an instrument requires reconfiguration to readdifferent wavebands or vessels the time taken to acquire a sequence ofreadings is inevitably increased (for example, movement of a filterwheel, or scanning of an optical system between wells will introduce aninevitable delay, and in any case if acquisitions are not concurrentthey will take longer). This has the effect of reducing the maximum rateof acquisitions, and hence reducing time resolution of measurements,which can be critical when the acquisitions are taken during a processsuch as a temperature ramp for the purposes of melt analysis.

It would therefore be useful to have a way of being able to distinguishand detect different wavelengths of light emanating from a number ofdifferent reaction vessels at the same time.

Accordingly, in a first aspect of the present invention there isprovided apparatus for detecting spectra in light emanating fromchemical or biochemical reactions occurring in at least one reactionvessel of a plurality of reaction vessels, each reaction vesselcomprising a receptacle portion having an emitting area from which lightcan emanate, said apparatus comprising a masking element having an arrayof small apertures through which light can pass, each small aperturebeing substantially smaller than the emitting area of the receptacleportion of the reaction vessel, there being one or more small aperturesarranged adjacent each of the reaction vessels, and a light detectingdevice for detecting the spectra in the light emanating from thechemical or biochemical reactions via the array of small aperturessubstantially simultaneously.

As used herein, the expression “reaction vessel” refers to any form ofsupport or container in which the reaction may be carried out. Thus, itincludes reaction tubes, wells in reaction plates as well as slides orchips.

Generally, the spectrum will be characteristic of a particular reagentsuch as a dye which is present in the chemical or biochemical reaction,and so the presence or absence, or intensity of the signal having thatcharacteristic spectrum may be indicative of a property or state of thereaction mixture.

As used herein, the expression “chemical or biochemical reaction”includes various operations in which reagents may react together inorder to produce new or different reagents or products, and also thetreatment of samples to determine the changes which take place inreagents under changing conditions, such as temperature, electrochemicalpotential or time. Thus the expression includes operations such asmelting point analysis of reagents, as well as reactions such as thePCR.

In one embodiment, the apparatus further comprises a light dispersingdevice for dispersing the light that escapes from the small apertures inthe masking element into a dispersed spectrum. The light dispersingdevice may be a light diverging device, such as a prism or a diffractiongrating.

The apparatus may further comprise a plurality of light waveguidesarranged to guide light from the small apertures in the masking elementto the light dispersing device.

In one embodiment, the light detecting device comprises a plane ontowhich the dispersed spectra of light from the apertures are produced,and one or more detectors for detecting specific spectra within thedispersed spectra.

The light dispersing device may comprise a light splitting device fordispersing the light into different wavebands.

The apparatus may comprise a plurality of light waveguides arranged toguide light from the small apertures in the masking element to the lightdetecting device.

In an embodiment, the masking element comprises at least two smallapertures per reaction vessel, each of the plurality of light waveguidesbeing arranged to guide light from a respective small aperture to thelight detecting device, wherein one waveguide per reaction vessel guidesthe light to one portion of the light detecting device for detecting onespecific spectrum of the light and another waveguide per reaction vesselguides the light to another portion of the light detecting device fordetecting one specific spectrum of the light.

In this embodiment, the different portions of the light detecting devicemay comprise light sensors sensitive to different spectra of the light.

Filters may be arranged between the light waveguides and the differentportions of the light detecting device, each respective filter passing adifferent spectrum of the light to the respective portion of the lightdetecting device.

Depending on the other elements of the apparatus, there may be otherbenefits—for example the simultaneous acquisition of each waveband fromthe or each vessel means that in systems where there may be fluctuationsin the excitation source, each waveband and vessel will be acquired atthe same excitation level. Any physical changes in the vessel, such asvessel movement or bubble formation, condensation or movement ofcontents will affect each waveband acquisition equally. This is aconsiderable benefit in cases where levels of one (often passive) dyeare used to normalise levels of another (often active) dye.

Since there is no need to alter the acquisition wavebands orvessel/detector alignment, for example by physical movement, thedetector can acquire data with minimal interruption. Since the detectorcan acquire all available wavebands just as easily as any subset ofwavebands, there is no need to work with a reduced waveband set, andthis increases opportunities for later analysis. Physical alignmentwithin the machine is also rendered less critical, since the detectoronly needs to be aligned to the vessels, rather than to external filtersetc., and any minor misalignment can be corrected for by processing ofthe detector image, for example pattern recognition and/or registrationmarks.

In order to generate a detectable signal from a chemical or biochemicalreaction, for example using fluorescent signaling reagents, it isfrequently necessary to illuminate the reaction mixture in order toprovide light energy, for example for the fluorophore to absorb, so asto allow it to emit light at its characteristic spectrum.

The signals may be monitored continuously or taken as certain particulartime points during each thermal cycle only, so that the changes overcycle number can be seen.

According to a second aspect, the invention provides an apparatus fordetecting spectra in light emanating from chemical or biochemicalreactions occurring in at least one reaction vessel of a plurality ofreaction vessels, each reaction vessel comprising a receptacle portionhaving an emitting area from which light can emanate, said apparatuscomprising a masking element having a small aperture adjacent eachreaction vessel through which light from that reaction vessel can pass,a plurality of light waveguides arranged to guide light from the smallapertures in the masking element to a light dispersing device fordispersing the light from each waveguide into a dispersed spectrum, anda light detecting device for detecting spectra in the dispersed spectraof light substantially simultaneously.

As mentioned above, the light dispersing device may comprise a prism ora diffraction grating.

The apparatus may further comprise an output array element having aplurality of output apertures arranged in a predetermined array adjacentthe light dispersing device, wherein each respective light waveguidecomprises a first end constrained to receive light from a respectivesmall aperture in the masking element and a second end constrained at arespective aperture in the array element to direct light to the lightdispersing device.

The light detecting device may comprise a plane onto which the dispersedspectrum of light from each aperture is produced, and one or moredetectors for detecting specific spectra within the dispersed spectra.The plane may be a sensing surface of the detector, or may be an imageplane on an optical element of the detector, which may contain suitableoptics to image the plane onto a sensing surface.

The array of output apertures in the output array element may bearranged so that the dispersed spectra on the plane of the lightdetecting device do not overlap, at least within the spectral rangewhere there is significant light emitted from the vessels and passed tothe sensor, and where the sensor has significant sensitivity. It will,of course, be apparent that the spectra could be considered to extendfrom deep UV to far infrared, and these wavelengths will overlap, butlight at these wavelengths can effectively be ignored where it is notexpected to be emitted, and/or the optics (e.g. prism) may well nottransmit it, and/or the sensor is not significantly sensitive to it.Even so, there may be filters over the sensor to block IR etc.—thismeans there is no effect if the IR portion of one spectrum overlapsanother spectrum, since the sensor won't detect the overlapped IR light.The arrangement may also be chosen to efficiently use the plane of thelight detecting device, for example to match its aspect ratio, andprovide only just enough space between the dispersed spectra tosubstantially prevent crosstalk between the spectra). Such anarrangement can improve the signal to noise ratio of measurements byproviding for readings across a greater area of the plane.

In one embodiment, the array of output apertures in the output arrayelement has a smaller area than the array of small aperturescorresponding to the array of reaction vessels.

The apparatus of any embodiment may also comprise a further lightwaveguide for each reaction vessel arranged between a further smallaperture in the masking element adjacent each reaction vessel and anexcitation light source for guiding light from the excitation lightsource to each of the reaction vessels.

There may be a plurality of excitation light sources, which may provideexcitation light of the same or different spectra, the excitation lightfrom each excitation light source being guided to each of the reactionvessels via one or more further light waveguides.

Thus, multiple excitation light sources may be provided, arranged sothat each source directs light into the further light waveguides.Alternatively, multiple further light waveguides may be provided to eachreaction vessel, each guiding excitation light from one or moreexcitation light sources.

Suitable excitation light sources include UV, Halogen, Xenon orfluorescent lamps, Light Emitting Diodes, Lasers, or some combination ofthese sources. This excitation causes fluorescent dyes or markers whichare contained with the reaction vessel to emit light with acharacteristic spectrum in the range of the spectrum suitable for thedetector type, and this can then be picked up by the detectors.

Excitation light sources are preferably restricted to regions of thespectrum that are distinct from the most informative emittedwavelengths, for example the peak emission wavelengths of anyfluorophores, reducing need for filtering and allowing use of a greaterportion of the spectrum without interference from reflected excitation.For example, ultraviolet and blue excitation light sources are usefulsince most commonly used fluorophores emit at longer wavelengths.

The channeling of multiple excitation light sources into the samewaveguide can be achieved for example by use of a dichroic mirrorarranged to transmit light from one light source positioned so as toemit light directly into the waveguide, and reflect light into thewaveguide from another source arranged to emit light perpendicular tothe waveguide.

Multiple sources of the same spectrum may be used to increase the powerof excitation light, or sources of different spectra may be used, forexample where each source is designed to provide acceptable excitationfor a specific set of fluorophores. Where multiple sources are provided,they may be individually controlled (in terms of intensity and spectrum)so that acquisitions may be made in the presence of a controlledexcitation spectrum. For example, a common application would be theacquisition of emitted fluorescence from FAM and VIC dyes, where a blueLED with appropriate filter provides excitation matched to the FAM dye,and a green LED with appropriate filter provides excitation matched tothe VIC dye. By illuminating just the blue LED when acquiring spectrafrom the FAM dye, a better reading can be made since the green LEDexcitation light will not be present to interfere with the FAM emissionat similar wavelengths. The green LED alone can then be illuminated toacquire a spectrum from the VIC dye.

The apparatus may also comprise one or more additional light waveguidesarranged to guide light from one or more excitation light sources to theoutput array element, without illuminating any reaction vessel. Such alight waveguide may also include a filter, for example a neutral densityfilter to reduce the intensity of light directed from the excitationlight sources to the output array element. These additional lightguidesprovide for the excitation sources to have their intensity and spectrameasured in the same way and at the same time as the light emitted andreflected from the reaction vessels. This provides for example forratiometric measurement, where the emitted light from the reactionvessels is compared to the spectrum and intensity of the excitationsource to yield a more accurate measurement having reduced influencefrom any variation in the excitation source intensity and spectrum.

The at least one reaction vessel is preferably formed in a generallytapered configuration and may be formed by a capillary.

Preferably, the emitting area is at a top of the receptacle portion,although it may be at a side of the receptacle portion and or at abottom of the receptacle portion.

The masking element may be provided by a thermal mount in which thearray of reaction vessels is mounted.

In another aspect, the invention provides an apparatus for detectingspectra in light emanating from chemical or biochemical reactionsoccurring in a plurality of reaction vessels of an array of reactionvessels, each reaction vessel comprising a receptacle portion having anemitting area from which light can emanate, said apparatus comprising aat least one light waveguide per reaction vessel being arranged to guidelight from the emitting area to a light dispersing device for dispersingthe light from the waveguide into a dispersed spectrum, and a lightdetecting device for detecting spectra in the dispersed spectra of lightsubstantially simultaneously, and at least one excitation arrangementfor providing excitation light to the receptacle portion of the reactionvessel.

Preferably, the excitation arrangement comprises a second lightwaveguide per reaction vessel for guiding excitation light from anexcitation light source to the receptacle portion of the reactionvessel.

The excitation arrangement preferably comprises an excitation lightsource arranged in or adjacent the receptacle portion of the reactionvessel.

The excitation light source preferably comprises a Light Emitting Diode(LED).

The light dispersing device may comprises a prism or a diffractiongrating.

Preferably, the emitting area is at a top of the receptacle portion,and/or at a side of the receptacle portion and/or at a bottom of thereceptacle portion.

The light detecting device may comprises a CCD or CMOS detector.

The plurality of reaction vessels may be contained within a multi-wellplate, such as a 48, 96 or 384 well plate.

The apparatus may further comprise a thermal cycler having a blockheater for holding the multi-well plate.

The specific spectrum may be characteristic of a particular reagent orstate of a particular reagent within a reaction vessel and/or may bederived from a single species of fluorophore, present in the reaction.

In a preferred embodiment, the chemical or biochemical reaction is apolymerase chain reaction (PCR) conducted in the presence of at leastone fluorophore, which may be one or more fluorophores taken from thegroup including:

-   -   fluorophores which intercalate with nucleic acid, such as        intercalating dyes;    -   fluoorophores which hybridize with nucleic acid, such as labeled        hybridization probes;    -   fluorophores which are modified by the PCR process, such as        labeled digestion probes;    -   fluorophores which provide for fluorescent energy transfer        between them, such as fluorescent labeled probes; and        other fluorescent probes.

The fluorophore is preferably a fluorescent label attached to a firstoligonucleotide probe which specifically hybridizes to a target nucleicacid sequence of the PCR and wherein the first oligonucleotide probecontains a second fluorophore, which is able to exchange fluorescentenergy with said fluorescent label when present together on the probe,wherein a polymerase having 5′-3′exonuclease activity is utilized in thePCR so as to digest any first probe bound to target nucleic acid duringan extension phase of the reaction.

If necessary, a cooling or refrigeration device may be provided forcooling the light detecting device, particularly when this is a CCD, toincrease signal to noise ratio and achieve more accurate readings.

Various embodiments of the invention will now be more fully described,by way of example, with reference to the accompanying diagrammaticdrawings, of which:

FIG. 1 shows a schematic diagram of a first embodiment of an opticalsystem for detecting light in a PCR system;

FIG. 2 shows a schematic diagram of a second embodiment of an opticalsystem for detecting light in a PCR system;

FIG. 3 shows a schematic diagram of a third embodiment of an opticalsystem for detecting light in a PCR system;

FIG. 4 shows a schematic diagram of a fourth embodiment of an opticalsystem for detecting light in a PCR system;

FIG. 5 shows a plan view of an array plate used in the embodiment ofFIG. 1;

FIG. 6 shows a plan view of an image area in the embodiment of FIG. 1;

FIG. 7 shows a schematic diagram, similar to FIG. 1, but includingexcitation in the optical system;

FIG. 8 shows a schematic diagram, similar to FIG. 1, but in analternative configuration;

FIG. 9 shows a schematic diagram, similar to FIG. 8, but includingexcitation in the optical system, similarly to the system of FIG. 7;

FIG. 10 shows a schematic diagram of another embodiment of an opticalsystem for detecting light in a PCR system;

FIG. 11 shows a schematic diagram, similar to FIG. 10, but includingexcitation in the optical system;

FIG. 12 shows a schematic diagram of a further embodiment of an opticalsystem for detecting light in a PCR system;

FIG. 13 shows a schematic diagram, similar to FIG. 12, but includingexcitation in the optical system;

FIG. 14 shows a schematic diagram of a still further embodiment of anoptical system for detecting light in a PCR system;

FIG. 15 shows a schematic diagram, similar to FIG. 14, but withoutseparate excitation LEDs;

FIG. 16 shows an enlarged view of part of the system of FIG. 13;

FIG. 17 shows a similar enlarged view to that of FIG. 16, but for partof the system of FIG. 15;

FIG. 18 shows a plan view corresponding to FIG. 17;

FIG. 19 shows a similar view to that of FIG. 17, but in an alternateconfiguration; and

FIG. 20 shows a plan view corresponding to FIG. 19.

Thus, turning first to FIG. 1, there is shown a multi-well array 1having a number of wells 2 in which are provided reaction vessels 3. Thearray 1 may well have any number of wells, for example, 48, 96 or 384 asin conventional such arrays. The array 1 may be housed in a heater block4 of a thermal cycler, as is well known in the field.

As will be apparent to a person skilled in this field, the reactionvessels 3, after having the desired reagents inserted therein, may besealed and may have a heated lid placed on it. The seal is usually oftransparent plastics material which is adhered to the rim of thereaction vessel and the heated lid, which is usually arranged so as toprovide pressure on the seal at the rim of the reaction vessel, andheated to reduce condensation on the inside of the seal is also usuallytransparent or provided with appropriate apertures to allow light fromthe reaction vessel to escape. These elements are not shown since theyare not part of the invention and are well known.

As shown in FIG. 1, a masking plate 5 is provided, which would bepositioned over the heated lid and/or seal, if present. The maskingplate prevents light from escaping from the reaction vessels 3, exceptfor through small apertures 6 positioned in the masking plate 5 so as tocorrespond to the approximate centre of each reaction vessel 3, therebyensuring that the maximum amount of light will impinge on the smallaperture 6. Inserted into each of the small apertures 6 is an opticalfibre 7, which guides the light emanating from the reaction vesselstowards a light dispersing element, such as a prism 8. One end of eachof the optical fibres 7 is mounted in or at the small aperture 6 and theother end is mounted in or at an aperture 30 provided in an array plate9, as shown in FIG. 5. It will be apparent that the optical fibres 7guide the light from each of the reaction vessels and direct it in apredetermined array towards the prism 8. The arrangement of thepredetermined array of apertures 30, as best shown in FIG. 5,effectively rearranges the array of light from a large array, which maybe roughly square in shape if the reaction vessels 3 have approximatelythe same width as length, into an array where the end of the fibres aremore closely packed together in one dimension (vertically in FIG. 5)than in the other direction (horizontally in FIG. 5). It is alsopossible to provide for the masking plate 5 to be heated, for example bypassing a current through resistive elements, and hence also function asa heated lid, if desired. In this case the fibres are preferably chosento be resistant to the temperatures required of the heated lid.

Thus, light from the ends of the optical fibres 7 in the array plate 9is directed along light path 11 to the prism 8 (or other lightdispersing element, such as a diffraction grating), which disperses thelight from each fibre 7 (and therefore from each reaction vessel 3) intoa full spectrum 12, as shown schematically in FIG. 1, into a detector10. The full spectra are imaged onto an image plane 13, as shown in FIG.6 in the detector 10. In this way, full spectra of the light emanatingfrom all the reaction vessels is provided simultaneously at the detector10. It will thus be seen that the spacing of the array in array plate 9is chosen so that spectra 12, when dispersed by the prism 8 onto theimage plane 13 in detector 10, are relatively tightly packed in onedirection, so that the height of the spectra are reasonable spaced, andare spaced sufficiently in the other direction so that the dispersedspectra do not overlap. Since the small apertures are substantiallysmaller in diameter that the size of the top area of the reactionvessels, the array of apertures in the array plate 9 (and the array offull spectra 12 in image plane 13) can be smaller than the size of thearray 1 of reaction vessels 3.

The detector 10 may, in one embodiment, consist of a ½″ (12 mm)monochrome CMOS sensor, together with appropriate electronics andsoftware allowing a “raw” frame to be captured giving the actualmeasured light levels for each pixel. This is used with a megapixelphotographic lens assembly to form a camera which can focus light from aplane in space onto the sensor chip. It should be noted that “lens” isused herein interchangeably to mean either an “optical lens”, a singlepiece of glass, or a “photographic lens”/“lens assembly” meaning one ormore lenses used as a set to image onto a sensor plane such as the CMOSsensor. The camera is then used to image through a simple single glasslens and a 30° uncoated glass prism onto the fibre array.

Sensors providing for global shutter control giving substantiallyequivalent exposure intervals for each pixel are well suited for usewith the system, since exposure of the entire image over the same timeperiod means that each channel of each spectrum in that image isaffected in the same way by any time varying conditions such as variableexcitation intensity, etc. For each reaction vessel, each channel isalso affected equally by any time varying conditions in the reactionvessel, such as condensation, temperature, physical movement such asbubble formation and movement etc.

Sensors that are well suited for use with the system include thoseproviding for different subsets of pixels across the sensor array to becaptured with different parameters, for example, electronic parameterssuch as analogue gain and offset, ADC reference voltage, pixel potentialbarrier, and other commonly controlled capture settings. Examplesinclude sensors such as the Micron MT9T001, where pixels are groupedinto 2×2 blocks, where the top left pixels of each block all belong toone subset, the top right pixels belong to another subset, and similarlyfor the bottom left and bottom right pixels. Each of these subsets ofpixels can have a different ADC gain parameter. This can be used toeffectively extend the dynamic range of the sensor; for example if again of 4× is used on even rows of the image, and a gain setting of 8×is used on odd rows, the spectral image will effectively be acquired astwo half resolution images with different gain levels, where the lowergain image has a higher maximum light level at saturation, and thehigher gain image provides greater precision at low light levels.Another example is the Aptina/Micron MT9V024 image sensor, where theimage can be divided into an array of rectangular regions, and eachrectangular region can have individual digital gain and gain controlsettings. The spectral image is particularly suitable for a sensorhaving different gain in different regions, since the regions can bearranged to coincide with the spectral images, giving different gainsettings for different areas of the spectra, and hence for differentwavelength regions. This can be used to acquire regions of the spectrathat have different intensity levels so as to give the best SNR andleast quantisation noise for each region.

Sensors providing a non-linear response in terms of output codes tolight level are well suited for use with the system, particularly wherethe sensor response can be programmed, for example by means of multiplelinear response regions and/or companding. An example of such a sensoris the Aptina/Micron MT9V024, which can use 12 bit to 10 bit companding,and can also be given up to 3 regions if different linear response,resulting in a greater dynamic range. For example, such sensors can beconfigured so that they yield higher light to output gain at low lightlevels, giving good SNR and sensitivity at the light levels associatedwith early cycle PCR amplification where measurement precision iscritical, but then yield lower gain at the higher light levelsassociated with mid and late cycle PCR in the plateau phase, wheremeasurement precision is less critical. A final region of even lowergain at very high light levels associated with reflection of theexcitation light can then be used to allow for measurement of thereflected light without the saturation that would result from a uniformhigher gain level.

As shown in FIG. 6, by providing a full spectrum 12 of dispersed lightfrom each reaction vessel at the same time, the detector 10 can detectany desired specific spectrum within the full spectrum. Thus, FIG. 6shows three wavebands (corresponding to the colours red 14, green 15 andblue 16) within a full spectrum 12, that can be detected, as desired. Ofcourse, particular wavelengths can also be detected, if desired, as canother wavebands. Each full spectrum 12 in the image plane 13 can bescanned by the detector and monitored and analysed, as required by timeand wavelength according to the requirements of the particular analysisbeing carried out, as will be apparent to a person skilled in the field.

In one alternative embodiment to that described above, the apertures 6need not be small relative to the area of the top of the reactionvessel, but can be made of substantially similar size thereto. In thiscase, the end of each of the optical fibres 7 that is mounted in or atthe aperture would be of substantially similar size and the diameter ofthe fibre would taper down to a smaller diameter, which would be that atthe other end mounted in or at aperture 30 provided in array plate 9. Itwill be apparent that this embodiment has the advantage thatsubstantially all the light emanating from the reaction vessels would becaptured by the large diameter end of the optical fibres and would thenbe “concentrated” as it passes through the tapering portion of thefibre. Between the array plate 9 and the detector 10, the system wouldbe the same as described above, so that both embodiments have theadvantage that the signals from the array of reaction vessels arerearranged into a format more suitable for passing through the prism andto the detector, i.e. that the overall size of the “image” passed fromthe array plate is smaller in overall size, than that of the array plateitself.

For example, in a direct image of the array of vessels taken from above,only about ¼ (or more) of the plate image area would normally have asubstantial amount of emitted light from the vessels—the remaining ¾ ofthe image is of the area between the vessels. When the masking elementand the fibres are placed between the array of vessels and the detectorand the emitted light is rearranged as explained above, the resultingimage is smaller than a direct image would be, allowing for the wholeimage (i.e. the light from all the vessels in the array) to be passedthrough the prism and on to the detector, even though less of theresulting image actually shows emitted light (for example, the systemmay actually have only about 1/10th of the area illuminated) so as toleave the necessary space for the well spectra to be dispersed withoutoverlapping.

Other embodiments of the invention will now be described, with the sameor similar elements as described above with respect to FIGS. 1, 5 and 6being given the same reference numbers. Thus, as shown in FIG. 2, asecond embodiment of the invention has the same elements as theembodiment of FIG. 1, except that the optical fibres 7 are not required.In this case, if the array 1 of reaction vessels 3 is not too large, itmay not be necessary to compress the array of full spectra imaged ontothe image plane 13 of the detector 10. In this case, however, themasking plate 5 is still used to block most of the light emanating fromthe reactions in the reaction vessels 3, and to only allow beams 11 oflight of much smaller diameter to pass through the small apertures 6 inthe masking plate 5 to the prism 8 and on to the image 13. This is sothat the full spectra 12 are prevented from overlapping on the imageplane 13, which would otherwise be the case if light from the completetop area of each reaction vessel 3 were to be dispersed by the prism.

The embodiments of FIGS. 3 and 4 similarly mask the reaction vessels 3using a masking plate 5 and only allow light to escape from eachreaction vessel via a small aperture 6 in the masking plate 5. Again, anoptical fibre 7 is mounted in or at the small aperture 6, but this time,there are several (in this case, three) such small apertures 6 providedadjacent each reaction vessel 3, so that there are several opticalfibres 7 per reaction vessel 3 guiding the light from each reactionvessel 3 to several separate portions of the detector 10. Here, thereare three such optical fibres 7 guiding the light from each reactionvessel 3 to three separate portions of the detector 10. The detector 10can thus be provided with separate portions for detecting differentspecific spectra, for example for detecting red, blue and green colours.Thus, no separate light dispersing element is needed.

As shown in FIG. 3, there are three sets 17, 18 and 19 of optical fibres7, the fibres within each set guiding light from each reaction vessel 3to different portions 20, 21, and 22 of the detector 10 for detectingred, green and blue specific spectra, respectively. Each respectiveportion of the detector 10 includes a sensor 23 and a filter 24, 25, and26. The second ends of the optical fibres of each set 17, 18 and 19 arearranged adjacent the respective filter 24, 25 and 26 so as to filterthe light from the second ends of the respective set of fibres so as tolimit the light reaching the respective sensor to a specific spectrum orwaveband. Thus, in this case, filter 24 is a red filter, filter 25 is agreen filter and filter 26 is a blue filter. The sensors 23 may be thesame or may be specific to the colour of light to be sensed by them.

Of course, if the sensors 23 are colour specific, so that they will onlydetect a specific spectrum, then the filters are not needed, as shown inFIG. 4, and the second ends of each set of optical fibres can bepositioned directly adjacent the appropriate one of the sensors, such asred sensor 27, green sensor 28 and blue sensor 29.

With most of the above described embodiments, it will be appreciatedthat another small aperture 37 can be position in the masking plate 5adjacent each reaction vessel 3 with one end of an optical fibre 31mounted in or at the small aperture 37, as shown in FIG. 7. Theseoptical fibres 31 can be used to bring excitation light to the reactionvessels by having their other ends positioned adjacent one or moresources of excitation light 32, 33. The excitation fibres 31 can bejoined together at the excitation accepting end, to make it easier todirect light into them. This may be a drilled plate 35, but this is notnecessary, since it is often easier just to bundle the fibres up into anapproximately hexagonally packed bundle.

In this embodiment (which is based on the first embodiment describedabove), one excitation light source may be a blue high intensity LED 32,having an asphere lens thereon. The other excitation light source may bea green LED 33. The LEDs 32 and 33 are arranged on either side of adichroic mirror 34 so as to combine the excitation light from both LEDs32 and 33 and to direct it to a homogeniser 36 (essentially a hexagonalprism or cylinder of glass). The dichroic mirror 34 allows blue lightfrom 32 to transmit, and reflects green light from LED 33 into thehomogeniser, which to gives more uniform illumination of each excitationlightguide, by reflecting the excitation light multiple times within thehomogeniser. This combination produces a spatially homogenousillumination of the polished end of the bundle of excitation fibres, sothat each reaction vessel 3 receives fairly equal excitation. It shouldbe noted that the dichroic mirror 34 could be replaced by some othermeans of directing light from both LEDs into the fibres, for example, aY shaped lightguide, or even just by having the LEDs angled to bothshine at an angle into the fibres.

One embodiment may have 16 pairs of emission/excitation fibres, mountedin cylindrical metal ferrules with one excitation and one emission perferrule. The ferrules can then be placed in the holes of a conventionalheated lid for use. The fibres are made from heat resistant plastics totolerate contact with the heated lid at ˜110 C.

Of course, if the tapered fibres are used, that cover substantially thewhole of the areas of the top of the reaction vessels, then there wouldbe no room for a further excitation fibre. In this case, the excitationlight can be guided by the same fibre that guides the emitted light.

In order, then, to detect the spectra from the reaction vessels, theexcitation source (blue or green) is turned on, left to settle for ashort time, and an acquisition is then made of an image of the fibreends. Various correction processes can be applied to this image; forexample, correcting for any offset in the reading by subtracting a“dark” image from the acquired image. This dark image is taken with thesensor exposed to as little light as possible, to measure the constantoffset that each pixel gives even without light (this is a standardoptical correction technique). A further processing stage is to discardpixels of the image which are considered not to be providing a reliablemeasure of light; for example, so-called hot pixels which give a higherreading due to current leakage or other manufacturing flaws.

The final corrected image then shows the spectra very much as depictedin FIG. 6. To correct for inevitable differences in the positioning ofthe optics and fibres, a calibration may be performed. This should benecessary only when the instrument has been first manufactured, or afterit has been disturbed—due to physical shock, disassembly, etc.Calibration may just use an empty vessel array to reflect the excitationlight back into each fibre. The relatively well defined image of thefibre ends in the image can then be seen, since the excitation light hasa narrow waveband. The location of each bright point for the reactionvessels can then be found either manually or automatically, and this canbe used as a fixed reference point in the spectrum for that reactionvessel. A rectangular (or other shaped) region for the spectrum of eachvessel is then defined and stored together with the calibration.

Finally, to interpret a given image, a spectrum is extracted for eachvessel. The spectral region for that vessel is looked up from thecalibration, and spectral area is then simply scanned along from left toright, averaging the intensity of the pixels in each area to give anintensity for the spectrum itself in the waveband corresponding to thosepixels. There are various means of converting, but a simple and adequateway is to average all the pixels in each vertical column of the spectralregion, giving more weight to the brighter pixels in the center of thespectrum vertically. Each column average then becomes the intensity forthat column, or channel of the reading. A final stage of correctionwould be to map the channels to the actual wavelength dispersed to thatcolumn of the image—this can be done by modelling the dispersingbehaviour of the prism or measuring known spectra, but may not always benecessary, since it is possible to compare spectra by channels ratherthan by wavelength.

Although in the above description, the light emanating from the reactionvessels has been shown as being emitted from an area at the top of thereaction vessel, it will, of course, be apparent that the emitting areacan be in any position. The “top” of the reaction vessel is intended tocover any position of the emitting area on the reaction vessel fromwhich the light emanates. Thus, for example, FIG. 8 shows the samesystem as FIG. 1, with the same elements having the same referencenumbers as in FIG. 1, but in a reversed configuration, where the maskingplate 5 is positioned adjacent the heater block 4, which, in this case,has holes 38 in the wells 2 between the main elements 39 of the heaterblock 4 exposing the reaction vessels 3. The reaction vessels 3 areformed in a generally tapered configuration so that the emitting areasof the reaction vessels 3 are at a lowermost point of the taperedreaction vessel. Of course, when the reaction vessels are sealed, thearray of reaction vessels in the heater block 4 can be arranged in anydesired configuration, so the lowermost point of the tapered reactionvessel 3 as shown in FIG. 8 may well be the “top” in the physical sense.

FIG. 9 shows the same system configuration as FIG. 8, but with theexcitation fibres 31 as in the system described above with respect toFIG. 7. In this case, as in FIG. 7, the masking plate 5 is provided witha second aperture 37 adjacent the hole 38 in the bottom of each well 2.As described above, the excitation fibres 31 guide excitation light fromexcitation light sources 32, 33 to the reaction vessels 2 to excite anyfluorophores therein.

FIGS. 10 and 11 illustrate similar embodiments to those of FIGS. 8 and9, but where the masking plate 5 is formed by the heater block 4 itself.As can be seen, in this case, the heater block elements 39 extendsubstantially below the wells 2 and provide the apertures 38 into whichthe fibres 7 (and 31 in the case of FIG. 11) are inserted. In theembodiment of FIG. 11, the fibres 7 and 31 are illustrated as combininginto one fibre before they are inserted into apertures 38. The fibres 7and 31 can be combined in any known way, for example by a form ofparallel combination, for example with multiple fibres being containedin the same outer jacket.

Turning now to FIG. 12, there is shown there an arrangement using anarray of capillary tubes 40 mounted in a mounting plate 41 in place ofthe reaction vessels 2. The capillary tubes 40 may be heated, forexample, by blowing heated gas around them. In this case, the remainingfeatures are similar to those of FIG. 10 and have the same referencenumerals, except that the ends of the fibres 7 protrude through themasking plate 5 to enable them to be positioned appropriately closely tothe end of the respective capillary tube 40. As best shown in FIG. 16,because a fibre 7 has a limited angle of reception 42 (or emission), byhaving the end of the fibre relatively close to the reaction fluid 43 inthe capillary means that any tolerances in the position of thecapillary, which may be somewhat greater in the case of the hangingcapillary tubes 40 than in wells in a heater block, are allowed for. Ofcourse, if the end of the fibre is too close, where the capillary endwill only just fit into the intersection of the excitation and emission“cones”, any horizontal movement of the capillary results in light notbeing captured. On the other hand, if the fibres are further away, suchas in the mounting plate 5, then variations in position, or movement ofthe capillary tubes 40 when in position, for example due to the pressureof the heating gas, would be allowed for due to the increase in the coneof reception 42, but there is a corresponding reduction in theefficiency of light collection and in excitation due to the increase insize of the excitation cone. As shown in the embodiment of FIG. 12,Light Emitting Diodes (LEDs) 44 of appropriate excitation light may beprovided adjacent each capillary tube 40. In the alternative embodimentshown in FIG. 13, the excitation light is provided in the same manner asin previous embodiments with excitation fibres 31 being used to bringexcitation light to the capillary tubes 40 from one or more sources ofexcitation light 32, 33.

FIG. 14 shows a still further embodiment, similar to that of FIG. 10,where the same elements have the same reference numerals. In this case,the excitation light is provided by LEDs 44, as in the embodiment ofFIG. 12. However, the holes 38 in the heater block 4 through which theends of the fibres 7 extend, are here positioned to extend upwardly fromthe base of the heater block and then to extend to the wells 2 in theheater block from a side thereof, so that the ends of the fibres 7 areadjacent the sides of the reaction vessels 3. It will, of course, beappreciated that the drawing does not show the full extent of the holes38, but only shows the fibres 7 extending upwardly through the heaterblock 4 in a schematic manner, with the hole 38 being shown adjacent theside of the well 2. The fibres 7 are also shown with a sharp right anglealthough, in practice, they would, of course, not be so sharply angled.As best shown in FIGS. 17 and 18, the fibres 7 need to be accuratelypositioned adjacent the side of the reaction vessel 3 so that the angleof reception 42 can capture as much light as possible emanating from thereaction fluid 43. However, as explained above, once in position, thereaction vessels 3 will not move unduly, so the results will not beaffected as much as with the capillary tubes 40.

FIG. 15 shows a further embodiment, similar to that of FIG. 14, in whichthe same elements have the same reference numerals. In this case, theholes 38 are made relatively small, with the ends of the fibres beingmounted within the holes so that the side of the well 2 in the heatingblock element 39 forms the masking plate to prevent light other thanthat passing through the aperture 38 from reaching the fibre 7. Althoughnot shown in FIG. 15, it will be appreciated that the excitation lightcould be provided by an excitation fibre 31 guiding excitation lightfrom one or more sources of excitation light, as in previousembodiments. Although the excitation fibre could be provided in the samehole 38 as the emission fibre 7, as shown in FIG. 17, this would meanthat neither the excitation fibre 31 nor the emission fibre 7 would bein the ideal position for the reaction vessel, since both the angle ofreception 42 and the angle of emission 45 would be off-centre. FIGS. 19and 20 show how the two fibres could be separated within the heaterblock so that their ends are adjacent the reaction vessel separated by90° so as to minimize the amount of excitation light that might enterthe emission fibre 7. As can be seen in FIG. 19, both the angle ofreception 42 and the angle of emission 45 are now centred on thereaction vessel 3, with reaction fluid 43 being wholly within the anglesof reception and emission of the respective fibres.

It will be appreciated that although only a few particular embodimentsof the invention have been described in detail, various modificationsand improvements can be made by a person skilled in the art withoutdeparting from the scope of the present invention. For example, it willprobably often be useful to have more optical components in the pathfrom plate 9 to plane 13 in FIG. 1. For example, it might be useful to“fold” the optical path 11 by adding one or more mirrors. This reducesthe size of the entire optical assembly, whilst making very littledifference the actual operation or performance of the system. Anotherexample of an additional component is to have one or more additionallenses before or after the prism 8 to provide additional correction ofthe light path—again this may not be essential, but is a well knownoptical technique to correct for various aberrations, etc. Suchadditional optical elements may form part of the dispersing element, butmay be separate components.

It will also be appreciated that, although the image plane 13 in FIG. 1is shown at the front of the detector 10, it could, alternatively, betowards the back of the detector, if the detector consists of a CMOSsensor with a suitable camera lens module in front of it. In theembodiments shown in FIGS. 14 and 15, the fibres pass vertically throughthe block 4, however, it will be appreciated that, in somecircumstances, the vertical portion of the fibres could be arranged tobe outside the block—for example in a block having one row of wells, thefibres could simply pass horizontally from the “side face” of the blockto the wells, since there are no other wells in the way. A mount withtwo rows of wells could obviously be arranged the same way, with fibresentering from the two longer vertical sides of the mount.

1. Apparatus for monitoring light emanating from at least one reactionvessel in which a chemical or biochemical reaction is occurring, saidapparatus comprising: an array of reaction vessels, each reaction vesselcomprising a receptacle portion in which a chemical or biochemicalreaction can occur, the receptacle portion having an emitting area fromwhich the light can emanate; a light detecting device for detecting oneor more wavebands of the light emanating from one or more of thereaction vessels; a plurality of light waveguides arranged to guide thelight from the emitting areas of the array of reaction vessels to thelight detecting device for detecting light emanating from one or more ofthe reaction vessels substantially simultaneously, each of the lightwaveguides having a first end arranged adjacent a corresponding emittingarea of a respective one of the reaction vessels, and a second end forproviding the light emanating from the respective reaction vessel to thelight detecting device, wherein the first ends of the plurality of lightwaveguides are arranged in a first array arranged adjacent the array ofreaction vessels, and the second ends of the plurality of lightwaveguides are arranged in a second array adjacent the light detectingdevice; at least one excitation light source for providing excitationlight; and a plurality of further light waveguides, each further lightwaveguide being arranged between a respective reaction vessel and the atleast one excitation light source for guiding excitation light from theat least one excitation light source to the respective reaction vessel.2. Apparatus according to claim 1, further comprising a plurality ofexcitation light sources providing excitation light of the same ordifferent spectra.
 3. Apparatus according to claim 1, where second endof each of the light waveguides is arranged adjacent a filter so as tolimit the light reaching the light detecting device to a specificspectrum or waveband.
 4. Apparatus according to claim 1, wherein thelight detecting device comprises a light dispersing device for producinga dispersed spectrum from the light emanating from each of the pluralityof reaction vessels on a plane, and one or more detectors for detectingspecific spectra within each of the dispersed spectra.
 5. Apparatusaccording to claim 1, wherein said first ends of the plurality of lightwaveguides are arranged in a plurality of apertures in a masking elementarranged adjacent the emitting areas of the reaction vessels. 6.Apparatus according to claim 5, wherein a first end of each furtherlight waveguide is arranged in a further aperture in the masking elementadjacent each reaction vessel.
 7. Apparatus according to claim 1,wherein the light waveguides taper in diameter from their first end,which has a diameter substantially similar to that of the emitting areaof the receptacle portion of the reaction vessel, whereby the second endof the light waveguides is substantially smaller in diameter than thefirst end.
 8. Apparatus according to claim 4, wherein the second arrayis arranged so that the dispersed spectra on a plane of the lightdetecting device do not overlap, at least within the spectral rangewhere there is significant light emitted from the reaction vessels andpassed to the detector, and where the detector has significantsensitivity.
 9. Apparatus according to claim 1, wherein the second arrayhas a smaller area than the first array.
 10. Apparatus according toclaim 5, wherein the masking element is provided by a thermal mount inwhich the array of reaction vessels is mounted.
 11. Apparatus accordingto claim 4, wherein the light dispersing device comprises a lightsplitting device for dispersing the light into different wavebands. 12.Apparatus according to claim 1, wherein the light emitting area is at atop of the receptacle portion of the reaction vessel.
 13. Apparatusaccording to claim 1, wherein the light emitting area is at a side ofthe receptacle portion of the reaction vessel.
 14. Apparatus accordingto claim 1, wherein the light emitting area is at a bottom of thereceptacle portion of the reaction vessel.
 15. Apparatus according toclaim 1, wherein the light detecting device comprises a CCD or CMOSdetector.
 16. Apparatus according to claim 1, wherein the array ofreaction vessels are contained within a multi-well plate.
 17. Apparatusaccording to claim 16, further comprising a thermal cycler having ablock heater for holding the multi-well plate.
 18. Apparatus accordingto claim 4, wherein the specific spectrum is characteristic of aparticular reagent or state of a particular reagent within a reactionvessel.
 19. Apparatus according to claim 4, wherein the specificspectrum is derived from a single species of fluorophore, present in thereaction.
 20. Apparatus according to claim 1, wherein the chemical orbiochemical reaction is a polymerase chain reaction (PCR) conducted inthe presence of at least one fluorophore, which may be one or morefluorophores taken from the group including: fluorophores whichintercalate with nucleic acid, such as intercalating dyes; fluoorophoreswhich hybridize with nucleic acid, such as labeled hybridization probes;fluorophores which are modified by the PCR process, such as labeleddigestion probes; fluorophores which provide for fluorescent energytransfer between them, such as fluorescent labeled probes; and otherfluorescent probes.
 21. Apparatus according to claim 20, wherein thefluorophore is a fluorescent label attached to a first oligonucleotideprobe which specifically hybridizes to a target nucleic acid sequence ofthe PCR and wherein the first oligonucleotide probe contains a secondfluorophore, which is able to exchange fluorescent energy with saidfluorescent label when present together on the probe, wherein apolymerase having 5′-3′exonuclease activity is utilized in the PCR so asto digest any first probe bound to target nucleic acid during anextension phase of the reaction.